Protective anti-chlamydial vaccine regimen-induced CD4+ T cell response mediates early inhibition of pathogenic CD8+ T cell response following genital challenge

Abstract We have demonstrated previously that TNF-α-producing CD8+ T cells mediate chlamydial pathogenesis, likely in an antigen (Ag)-specific fashion. Here we hypothesize that inhibition of Ag-specific CD8+ T cell response after immunization and/or challenge would correlate with protection against oviduct pathology induced by a protective vaccine regimen. Intranasal (i.n.) live chlamydial elementary body (EB), intramuscular (i.m.) live EB, or i.n. irrelevant antigen, bovine serum albumin (BSA), immunized animals induced near-total protection, 50% protection, or no protection, respectively against oviduct pathology following i.vag. C. muridarum challenge. In these models, we evaluated Ag-specific CD8+ T cell cytokine response at various time-periods after immunization or challenge. The results show protective efficacy of vaccine regimens correlated with reduction of Ag-specific CD8+ T cell TNF-α responses following i.vag. chlamydial challenge, not after immunization. Depletion of CD4+ T cells abrogated, whereas adoptive transfer of Ag-specific CD4+ T cells induced the significant reduction of Ag-specific CD8+ T cell TNF-α response after chlamydial challenge. In conclusion, protective anti-chlamydial vaccine regimens induce Ag-specific CD4+ T cell response that mediate early inhibition of pathogenic CD8+ T cell response following challenge and may serve as a predictive biomarker of protection against Chlamydia -induced chronic pathologies.


Introduction
Chlam ydia trac homatis is the leading cause of sexuall y tr ansmitted bacterial disease worldwide.Significant efforts are being made to de v elop a v accine to pr e v ent female r epr oductiv e pathology.We and others have demonstrated previously that IFN-γ -producing Ag-specific CD4 + T cell response induced by vaccination regimens is necessary, and a sufficient source of IFN-γ production, to induce pr otection a gainst r epr oductiv e pathology in naïve recipient mice that are challenged with C. muridarum (Morrison and Morrison 2001, Li et al. 2008, Gondek et al. 2012 ).We and others have also identified pr e viousl y a number of chlamydial antigens as putative vaccine candidates (Pal et al. 2001, Murthy et al. 2007, Murthy et al. 2011, Yu et al. 2012, Yu et al. 2016 ).Whereas imm unization with man y c hlamydial antigens induces a robust Th1 Ag-specific CD4 + T cell r esponse, onl y a subset of regimens induces early resolution of infection and/or significant reduction of c hr onic r epr oductiv e tr act pathology (Y u et al. 2016 ).Furthermor e, m ulti-functional CD4 + T cells that produce TNF-α along with IFN-γ have been shown to correlate better with earl y r e-duction in bacterial shedding when compared to regimens that induce pr edominantl y IFN-γ -pr oducing CD4 + T cells (Yu et al. 2016 ).Ho w e v er, r egimens that induce early reduction of bacterial shedding do not always induce reduction of c hr onic r epr oductiv e tr act pathology, and conv ersel y some r egimens that significantl y r educe c hr onic r epr oductiv e tr act pathology do not induce earl y reduction of chlam ydial shed ding (Andrew et al. 2013, O'Meara et al. 2014 ).As suc h, imm une corr elates that accur atel y pr edict pr otectiv e imm unity a gainst upper r epr oductiv e pathology, the prime reason that necessitates an anti-Chlamydia vaccine , ha ve yet to be defined.
To this end, we have demonstrated previously that TNF-αproducing CD8 + T cells cause chlam ydial re producti ve pathologies (Murthy et al. 2011 ).In ad dition, TNF rece ptor 2 on CD8 + T cells and TNF receptor 1 on non-CD8 + T cells contribute significantl y to c hlamydial pathogenesis (Manam et al. 2015 ).Importantly, CD8 + T cells with specificities to non-chlamydial antigens do not induce r epr oductiv e pathology following genital chlamydial infection (Manam et al. 2013, Vlcek et al. 2016 ), suggesting that antigen (Ag)-specific CD8 + T cells mediate chlamydial pathogenesis.As such, we hypothesized that the reduction/inhibition of Ag-specific CD8 + T cell TNF-α response after immunization and/or challenge would correlate with the pr otectiv e efficacy of a v accine r egimen a gainst c hr onic r epr oductiv e pathology.In addition to TNF-α response, we evaluated the production of two other cytokines as activation markers of Ag-specific CD8 + T cells, .Furthermore, we elucidated the effects of pr otectiv e CD4 + T cells on suc h earl y inhibition of Ag-specific CD8 + T cell response following genital chlamydial challenge in vaccinated animals.

Materials and methods
An ov ervie w of the experimental sc heme used in this manuscript is shown in Fig. 1 .

Chlamydia muridarum and mice
Chlamydia muridarum Nigg ( C. muridarum ) strain w as gro wn in HeLa 229 cells, and elementary bodies (EB) were obtained as described pr e viousl y (Murthy et al. 2007 ).Female four-to six-weekold C57BL/6 J WT mice w ere pur chased from the Jackson Laboratory and maintained at Mid western Uni versity.Food and water were supplied ad libitum and all experiments described in this manuscript wer e a ppr ov ed by the Institutional Animal Care and Use Committee at Midwestern University.

Intr av aginal infection of mice, monitoring of bacterial shedding, and estimation of upper reproducti v e tract pathology
On day 60 after imm unization, mice wer e c hallenged with 5 × 10 4 inclusion-forming units (IFU) of C. muridarum contained in 10 μl of sucrose-phosphate-glutamate (SPG) buffer placed into the vaginal vault.Mice were treated with 2.5 mg of Depo-pr ov er a ® (Pfizer, New York, NY) per mouse subcutaneously 5 days before vaginal challenge to induce anestrous and rece pti ve to the genital infection.The course of infection was followed by swabbing the v a ginal vault on the indicated days following inoculation (BSA i.n., n = 7; live EB i.m; n = 8, and live EB i.n., n = 8).Chlamydial counts were determined separately for each mouse as described previously (Murthy et al. 2007 ).Experiments were repeated twice and r esults fr om individual experiments anal yzed independentl y.
The upper genital tract pathology in mice infected with C. muridarum was also e v aluated on day 80 post-challenge, as described pr e viousl y (Murthy et al. 2011 ).Groups of mice were euthanized, and genital tracts removed (BSA i.n., n = 14; live EB i.m., n = 15, and live EB i.n., n = 17) for pathology analysis.Results from two experiments were pooled.

Splenic chlamydia -specific CD8 + T cell cytokine response after immunization or chlamydial genital challenge in immunized animals
On day 7, 14, or 21 following immunizations, or on day 3, 6, 14, or 21 after C. muridarum challenge, mice ( n = 4-5 per time-period) were euthanized, spleens collected and pooled single cell suspensions for each group were prepared.CD8 + T cells were enriched using negative selection magnetic beads (Stemcell technologies , C A). Enriched CD8 + T cells (1 × 10 6 cells/well, > 95% pure, Supplementary Fig. S1 ) were cultured with equal number of mouse antigen presenting cells pre-infected with live C. muridarum (MOI = 1) or incubated with control antigens.At the end of 72 hr incubation, supernatants were collected to analyze cytokine production.

CD4 + T cell depletion and monitoring
Groups of C57BL/6 mice ( n = 20) were immunized with 20 μL of live EB i.n.(2 × 10 3 IFU/mouse).At day 60 after immunization, mice c hallenged i.v a g. with Chlam ydia and wer e injected intr av enousl y with 150 μg/mouse of anti-CD4 depleting antibodies (GK1.5 clone, Bio X Cell, Lebanon, NH) or r at IgG2b isotype contr ol imm unoglobulin (Bio X Cell) in 200 μl of sterile 1X PBS, e v ery third day over a 15day period.Some spleens were collected and analyzed using flow cytometry to confirm the CD4 depletion ( Supplementary Fig. S2 ).Mice ( n = 4-5 per time-period) were euthanized on day 9 and 12 following intr av a ginal c hallenge.

Sta tistical anal yses
Comparisons of two groups was conducted using Student's t test, wher eas anal ysis of v ariance (One-wa y ANOVA; Systat, C A) was used for all comparisons of multiple groups .T he variance between gr oups compar ed statisticall y was similar.The differ ences in incidence of oviduct pathology were compared between two groups at a time using Fisher's exact test.Linear r egr ession anal-Figur e 1. Experimental scheme .T his dia gr am shows the v arious interv entions and anal yses conducted in this man uscript, along with the n umber of mice included in each experiment.
yses were conducted to determine if the level of cytokine production could predict chlamydial shedding and/or pathology as measured by the diameter of the oviduct in mice.The relationship betw een c ytokine pr oduction after imm unization or c hallenge to the shedding of Chlamydia after challenge (average of averages) or oviduct pathology (av er a ge diameter in the group) was assessed b y P earson's corr elation.Differ ences between gr oups wer e consider ed statisticall y significant if p v alues wer e ≤ 0.05.All experiments wer e r epeated at least twice, and eac h experiment was anal yzed independentl y, except for upper genital tract pathology r esults wher ein r esults fr om tw o experiments w ere pooled and analyzed.

Platform for analysis of immune correlates of protection
We compared the protective efficacy of three vaccine regimens a gainst v a ginal c hlam ydial shed ding and oviduct pathology.Mice immunized with BSA i.n.displayed high titers of vaginal chlamydial shedding early, follo w ed b y a pr ogr essiv e r eduction and cessation of shedding by day 30 after challenge (Fig. 2 A).Live Chlamydia elementary body (EB) i.n.immunization induced dramatic reduction in shedding as early as day 3 after challenge, and induced cessation of shedding by day 12 after challenge, as shown pr e viousl y (Li et al. 2010 ).Live EB i.m. immunization displayed comparable v a ginal c hlam ydial shed ding to BSA i.n. until day 9, follo w ed b y significant reduction in shedding from days 12 to 21, and cessation of shedding by day 24 after challenge.We also measured the incidence (Fig. 2 B) and se v erity (Fig. 2 C) of oviduct pathology on day 80 after challenge.We found that live EB i.n.immunization induced near-total pr otection, liv e EB i.m. imm unization induced 50% pr otection, and irr ele v ant antigen (BSA i.n.) imm unized animals displayed no protection against oviduct pathology following i.v a g. C. muridarum c hallenge (Fig. 2 A).Furthermor e, the se v erity of oviduct pathology was significantly reduced in live EB i.n. and live EB i.m. imm unized mice, when compar ed to BSA i.n.imm unized animals .T hese results were expected as we and others have previousl y demonstr ated the pr otectiv e efficacies of these regimens individually in different experimental contexts (Murthy et al. 2011, Lu et al. 2012 ).These models served as a platform of near-total pr otection, moder ate pr otection, and no pr otection a gainst v a gi-nal chlam ydial shed ding and oviduct pathology in whic h imm une corr elates wer e further c har acterized.

Total cellular cytokine response after immunization
Cellular IFN-γ , TNF-α, and IL-17 r esponse after imm unization was analyzed on day 7, 14, and 21 after initial immunization in single cell suspensions of spleen and draining lymph nodes (DLN).As shown in Fig. 3 A, liv e EB i.n.imm unization and liv e EB i.m. immunization both induced robust Ag-specific IFN-γ , TNF-α, and IL-17 in DLN cells on day 7, 14 and 21 after immunization.A similar response was also found in the splenocytes (Fig. 3 B).Splenocytes and draining lymph node cells from BSA i.n.immunized animals displayed minimal Ag ( Chlamydia )-specific cytokine response .T hese results demonstrated that an antigen-specific cellular immune response was induced as expected in the three groups of immunized animals.

Splenic CD8 + T cell response after immunization
We further e v aluated the splenic Chlam ydia -specific IFN-γ , TNFα, and IL-17 CD8 + T cell response in the three groups of animals on day 7, 14, and 21 following immunization (Fig. 4 ).As expected, the response in BSA i.n.immunized mice was minimal.Splenic CD8 + T cells from live EB i.n. and live EB i.m. immunized mice displayed robust Chlamydia -specific IFN-γ , TNF-α, and IL-17 response at all e v aluated time-periods.Live EB i.n.immunized mice displayed significantly enhanced CD8 + T cell TNF-α response on day 7 and IL-17 response on day 14 after immunization.Howe v er, Pearson co-efficient did not indicate a significant correlation between these ele v ated r esponses and either enhancement of v a ginal c hlam ydial shed ding or reduction of oviduct pathology.Ther efor e, the measur ed Ag-specific CD8 + T cell r esponses after imm unization wer e not r eflectiv e of the pr otectiv e ca pability, or lac k ther eof, of the e v aluated anti-c hlamydial v accine r egimens.

Splenic CD8 + T cell response following immunization and vaginal chlamydial challenge
The correlation of protective immunity to splenic Ag-specific CD8 + T cell responses at day 3, 6, 9, or 12 following intr av a ginal challenge was evaluated in the three groups of immunized animals.As shown in Fig. 5 A, BSA i.n.immunized and Chlamydiachallenged mice displayed minimal Ag-specific CD8 T cell IFN-γ response on day 3, peak response on day 6, and maintained ro- bust response on days 9 and 12 after challenge .T he Ag-specific CD8 + IFN-γ response of live EB i.n. or live EB i.m. immunized mice was significantly lower when compared to BSA i.n. on days 9 or12 after c hallenge.Mor eov er, Ag-specific CD8 + T cell IFN-γ response on day 9 after challenge in liv e EB i.n.imm unized mice is significantly lo w er when compar ed to liv e EB i.m. imm unized animals.
BSA i.n.imm unized and Chlam ydia -c hallenged mice displayed minimal Ag-specific CD8 + T cell TNF-α response on day 3, peak response on day 6, and maintained robust response on days 9 and 12 after c hallenge.Inter estingl y, the Ag-specific CD8 + TNF-α r esponse attained a similar peak in live EB i.n. or i.m. immunized animals as the BSA i.n.immunized animals on day 6 after challenge .T he Ag-specific CD8 + TNF-α response of live EB i.n. was significantl y r educed compar ed to BSA i.n. on days 9 or 12 after challenge, with a minimal response in live EB i.n.immunized animals on day 12 after challenge.The response in live EB i.m. immunized animals was significantly reduced compared to BSA i.n. on day 12 after c hallenge, and liv e EB i.n.imm unized mice displayed further significant r eduction compar ed to liv e EB i.m. animals on day 9 after challenge.
BSA i.n.imm unized and Chlam ydia -c hallenged mice displayed minimal Ag-specific CD8 + T cell IL-17 response on day 3, peak response on day 6, and maintained robust response on days 9 and 12 after challenge .T he IL-17 response in live EB i.m. immunized animals was comparable to BSA i.n. at all e v aluated timeperiods, whereas that in live EB i.n.immunized mice was significantl y enhanced compar ed to either BSA i.n. or liv e EB i.m. im-munized animals.Pearson's coefficient analyses indicated that onl y the r eduction in Ag-specific CD8 + T cell TNF-α r esponse on day 12 after c hallenge corr elated significantl y (r 2 = 0.99) with reduction in oviduct pathology, (Fig. 5 B).Collectively, these results suggest that Ag-specific CD8 + T cell TNF-α response at day 12 after v a ginal c hlamydial c hallenge corr elates significantl y with the pr otectiv e efficacy of vaccine regimen against oviduct pathology.

Role of CD4 + T cells in vaccine-induced reduction of pathogenic CD8 T cell response after challenge
Most successful vaccine regimens described in literature rely on the induction of Th1 type Ag-specific CD4 + T cell response in order to induce pr otectiv e imm unity a gainst genital c hlamydial infection and pathology (r e vie wed in (Yu et al. 2016 )).We hypothesized that depletion of CD4 + T cells after challenge would abrogate the inhibition of splenic Ag-specific CD8 + T cell TNF-α response at day 9 and/or 12 after challenge.Mice immunized with live EB i.n.wer e tr eated either with an anti-CD4 neutr alizing/deleting antibody or with the rat IgG2b isotype contr ol imm unoglobulin after challenge.As shown in Fig. 6 , Ag-specific CD8 + T cells from the mice depleted of CD4 T cells displayed a significant enhancement in IFN-γ response on day 9 and 10, TNF-α on day 12, and significant reduction of IL-17 on day 9 and 12 after challenge .T hese r esults demonstr ate that CD4 + T cells ar e necessary to inhibit the Ag-specific CD8 + T cell-TNF-α response that induces pathology following genital chlamydial infection.We further e v aluated the sufficiency of Ag-specific CD4 + T cells in naïve recipient mice to mediate inhibition of Ag-specific CD8 + T cell-TNF-α response in mice after genital chlamydial challenge.CD4 + T cells fr om liv e EB i.n.imm unized or moc k-imm unized animals were adoptively transferred into recipient unimmunized mice that were challenged i.vag.with C. muridarum .As shown in Fig. 7 , Ag-specific CD8 + T cells from mice receiving Ag-specific CD4 + T cells displayed a significant reduction of Ag-specific CD8 T cell IFN-γ , TNF-α, and IL-17 response on day 12 after challenge, when compared to mice receiving CD4 + T cells from mockimmunized animals .T hese results suggest that Ag-specific CD4 + T cells induced by pr otectiv e v accine r egimens ar e sufficient, in an otherwise untrained adaptive immune system environment, to mediate inhibition of an evolving pathogenic Ag-specific CD8 + T cell response following genital challenge.

Discussion
A licensed product has yet to be attained despite several decades of r esearc h into the de v elopment of an anti-Chlam ydia v accine.Multiple studies using various whole or subunit vaccines have demonstr ated that man y r egimens can successfull y induce r obust   Manam et al. 2015, Vlcek et al. 2016 ).In this study, we validated the ov er arc hing hypothesis that pr otectiv e CD4 + T cell r esponses induced after vaccination inhibit pathogenic CD8 + T cell response following genital chlamydial challenge.
CD8 + T cell response induces protective immunity against a number of other intracellular pathogens and such effects have been explored in Chlamydia infections (reviewed in (Brunham and Rey-Ladino 2005 )).Despite nearly three decades of research along these lines, evidence for the role of CD8 + T cells in anti-chlamydial pr otectiv e imm unity, whether induced after natur al infection or upon vaccination, is modest (reviewed in (Yu et al. 2016, Murthy et al. 2018 )).One pr obable r eason is that c hlamydial or ganisms ar e lar gel y localized to an endosomal compartment, but not in cytosol, during the de v elopmental cycle (r e vie wed in (Abdelr ahman and Belland 2005 )).And also Chlamydia -infected cells have been shown to e v ade CD8 + T cell-mediated r ecognition and killing of infected cells (Ibana et al. 2011 ).Conv ersel y, an isolated nonhuman primate study using the ocular tr ac homa model demonstrated that memory CD8 + T cells induced after multiple infectious challenges with a li ve-atten uated strain of Chlamydia contributed to solid pr otectiv e imm unity a gainst further r e-c hallenge (Oliv ar es-Zav aleta et al. 2014 ).Certain clones of CD8 + T cells (Igietseme et al. 1994 ), especially those exhibiting specific phenotypes (J ohnson et al. 2014 ), ha v e been demonstr ated to induce pr otectiv e imm unity a gainst genital c hlamydial c hallenge (Jiang et al. 2013, Johnson et al. 2020 ).One clone has been shown to possess specificity against a chlamydial protein that accesses the MHC-I pr esentation pathway earl y in the de v elopmental cycle (Starnbach et al. 2003 ), possibly before the immune evasion mechanisms are induced in the infected cell.On the other hand, we have shown that depletion of the polyclonal CD8 + T cells results in a reduction of pathology, without affecting chlamydial clearance (Murthy et al. 2011 ).Mor eov er, r epletion of TNF-α + / + CD8 + T cells in CD8 gene deficient mice r estor es the pathology comparable to wild type animals (Murthy et al. 2011 ).Consistently, Guangming et al. (Xie et al. 2020 ) demonstrated that CD8 + T cells from OT1 mice also significantly inhibited hydrosalpinx development in wild-type mice following an intr av a ginal inoculation with Chlam ydia.The y conclude the hydrosalpinx-inhibitory CD8 + T cells are Chlamydia nonspecific or independent of chlamydial antigen recognition.As such, the predominant role of CD8 + T cells during natural chlamydial infection, at least in the mouse model, appears to be pathogenic, although certain clones and phenotypes of CD8 + T cells may be harnessed via vaccine platforms to induce pr otectiv e imm unity.Consequentl y, a concern was whether induction of Ag-specific CD8 + T cell response upon vaccination would be detrimental.Our results suggest that Agspecific CD8 T cell responses induced upon immunization do not corr elate significantl y with pathological outcomes after challenge.Whereas we cannot extrapolate findings in mice directly to human individuals, our results suggest that vaccine-induced Agspecific CD8 + T cell responses per se may not be deleterious and support the continued exploration of ways to harness vaccineinduced CD8 + T cell responses, or a subset thereof, to induce pr otectiv e imm unity a gainst genital c hlamydial infection and/or pathology.
Notwithstanding the abo ve , we explored the correlation of v accine-induce pr otection a gainst c hlam ydial shed ding and/or oviduct pathology to Chlamydia -specific CD8 + T cell response after genital chlamydial challenge.Interestingly, our results show that a comparable activation of peak Ag-specific CD8 + T cell response occurred in all groups of animals initially after challenge.Ho w e v er, a significant correlation of subsequent inhibition of Chlam ydia -specific TNF-α pr oduction fr om CD8 + T cells , on da y 12 after challenge, was found in animals receiving protective vaccine regimens .T his suggested that the pathogenic CD8 + T cell pr ogr am was initiated in all challenged animals but was sub v erted by a protectiv e imm une r esponse quic kl y.The timing of suc h inhibition w as betw een 6 and 12 days and correlated with the pr e viousl y published timeline of 3-6 days (Roan et al. 2006, Li et al. 2008 ) for induction of pr otectiv e CD4 + T cell r esponse in c hallenged animals (r e vie wed in (Yu et al. 2016 )).Furthermore, we found that depletion of CD4 + T cells results in enhanced Ag-specific CD8 + T cell TNF-α response on day 12 after challenge .Con versely, adoptiv el y tr ansferr ed CD4 + T cells induced a significant r eduction of Ag-specific CD8 + T cell TNF-α response at the same time-period.In addition, the optimized OT1 mouse model study also sho w ed that the adoptive transfer of Chlamydia -primed CD4 + T cells failed to promote chlamydial induction of pathology in OT1 mice (Zhou et al. 2022 ).These results demonstrate that Ag-specific CD4 + T cells may be necessary and sufficient, in an otherwise untrained ada ptiv e imm une envir onment, to quic kl y inhibit the pathogenic CD8 + T cell TNF-α response cascade, and subsequently prevent or r educe c hr onic upper r epr oductiv e tr act pathology.The inhibition of the Ag-specific CD8 + T cell TNF-α response following challenge may be due to the excess pro-inflammatory cytokines including IL-12 and IFN-γ production from Ag-specific CD4 + T cells (Zhang and Starnbach 2015 ).
In summary, the r esults fr om these studies suggest that the early inhibition of Chlamydia -specific CD8 + T cell TNF-α response may be a mechanism by which protective CD4 + T cell responses pr otect a gainst upper r epr oductiv e tr act pathology following genital chlamydial infection.Furthermore, such information indicates the potential utility of Ag-specific CD8 + T cell TNF-α response as an earl y pr edictiv e biomarker of pr otectiv e efficacy of v accine r egimens in future clinical trials.

Figure 2 .
Figure 2. Platform for analysis of immune correlates of protection.Groups ( n = 7-8) of C57BL/6 J mice were immunized intranasally with irrelevant antigen, BSA, or live chlamydial EB, or intramuscularly with live chlamydial EB on day 0. On day 60, mice were challenged i.vag with C. muridarum .(A) Mean ± SEM of v a ginal c hlam ydial shed ding at indicated time-periods is sho wn.Significant ( P ≤ 0.05; ANOVA) difference betw een * liv e EB i.n.v ersus BSA i.n., * * live EB i.m. versus BSA i.n., * * * i.n live EB versus i.m. live EB.On day 80 following challenge , o viduct pathology was evaluated.(B) P er centage of mice displaying hydrosalpinx is shown.Significant ( P ≤ 0.05; Fisher's exact test) difference between * live EB i.n.versus BSA i.n., * * live EB i.m. versus BSA i.n., * * * i.n live EB versus i.m. live EB.(C) the incidence of dilated oviducts and mean ± SEM of oviduct diameter in each group is shown.Each mark er re presents an individual o viduct.T he horizontal line distinguishes normal from dilated o viducts .T he number of dilated oviducts (numerator) and total number of oviducts examined (denominator) for each group is also shown.Significant ( P ≤ 0.05; Fisher's exact test) difference between * live EB i.n.versus BSA i.n., * * live EB i.m. versus BSA i.n., * * * i.n live EB versus i.m. live EB.All experiments were repeated twice, and composite results are shown.

Figure 3 .
Figure 3.Total cellular antigen-specific cytokine response after immunization.Groups ( n = 15) of C57BL/6 J mice were immunized intranasally with irr ele v ant antigen, BSA, or liv e c hlamydial EB, or intr am uscularl y with liv e c hlamydial EB on da y 0. On da y 7, 14, or 21 after immunization, mice ( n = 4-5) were euthanized.Lymphocytes from draining iliac or mediastinal lymph nodes (A) or splenocytes (B) were stimulated in vitro with UV-irr adiated c hlamydial EB.Mean ± SEM of IFN-γ , TNF-α, or IL-17 production is shown.Significant ( P ≤ 0.05; ANOVA) differ ence between * liv e EB i.n.versus BSA i.n., * * live EB i.m. versus BSA i.n., * * * i.n live EB versus i.m. live EB.Results from individual experiments are shown and were analyzed independently.All experiments were repeated twice to confirm the findings.

Figure 4 .
Figure 4. Splenic CD8 + T cell-antigen-specific cytokine response after immunization.Groups ( n = 15) of C57BL/6 J mice were immunized intranasally with irr ele v ant antigen, BSA, or liv e c hlamydial EB, or intr am uscularl y with liv e c hlamydial EB on da y 0. On da y 7, 14, or 21 after immunization, mice ( n = 4-5) were euthanized and CD8 + T cells were purified from splenocytes.Purified CD8 + T cells were stimulated in vitro with live chlamydial EB-infected antigen presenting cells.Mean ± SEM of IFN-γ , TNF-α, or IL-17 production is shown.Significant ( P ≤ 0.05; ANOVA) difference between * live EB i.n.versus BSA i.n., * * live EB i.m. versus BSA i.n., * * * i.n live EB versus i.m. live EB.Results from individual experiments are shown and were analyzed independently.All experiments were repeated twice to confirm the findings.

Figure 5 .
Figure 5. Splenic CD8 + T cell-antigen-specific cytokine response after challenge.A. Groups ( n = 20) of C57BL/6 J mice were immunized intranasally with irr ele v ant antigen, BSA, or liv e c hlamydial EB, or intr am uscularl y with liv e c hlamydial EB on da y 0. On da y 60, mice wer e c hallenged i.v a g. with C. muridarum .On day 3, 6, 9, or 12 after challenge, mice ( n = 4-5) were euthanized and CD8 + T cells were purified from splenocytes.Purified CD8 + T cells wer e stim ulated in vitro with liv e c hlamydial EB-infected antigen pr esenting cells.Mean ± SEM of IFN-γ , TNF-α, or IL-17 production is shown.Significant ( P ≤ 0.05; ANOVA) difference between * live EB i.n.versus BSA i.n., * * live EB i.m. versus BSA i.n., * * * i.n live EB versus i.m. live EB.Results from individual experiments are shown and were analyzed independently.B .Pearson' s correlation co-efficient between oviduct diameter to Ag-specific TNF-α pr oduction fr om splenic CD8 + T cells was anal yzed.All experiments wer e r epeated twice to confirm the findings.

Figure 6 .Figure 7 .
Figure6.Effect of CD4 + T cells on splenic CD8 + T cell-antigen-specific cytokine response after challenge in immunized animals.Groups ( n = 10) of C57BL/6 J mice were immunized intranasally with live chlamydial EB on day 0. On day 60, mice were challenged i.vag.with C. muridarum and treated with anti-CD4 antibody ( n = 5) or rat IgG2b isotype control immunoglobulin ( n = 5).On day 9 or 12 after the challenge, mice were euthanized and CD8 + T cells were purified from splenocytes.Purified CD8 + T cells were stimulated in vitro with live chlamydial EB-infected antigen presenting cells.Mean ± SEM of IFN-γ , TNF-α, or IL-17 production is shown.* Significant ( P ≤ 0.05; Student's t test) difference between the groups.Results from individual experiments are shown and analyzed independently.All experiments were repeated twice to confirm the findings.